It has been clearly established in both clinical and experimental models that diaphragm disuse as a result of placement upon full mechanical ventilator support leads to early and severe diaphragm atrophy and dysfunction (VIDD - Ventilator-Induced Diaphragm Dysfunction). This diaphragm dysfunction appears to be an important cause of the difficulty often encountered weaning patients from mechanical ventilation (MV); it is thus the basis for subsequent ventilator-associated complications which result in increased morbidity, mortality and health care costs among the millions of patients requiring MV yearly. Several important mechanisms underlying VIDD have been elucidated in recent years, with mitochondrial oxidative stress emerging as a central effector of the process, but preclinical therapeutic trials based upon these mechanistic insights are only now beginning. The broad goals of this work are to: 1) Carry out preclinical trials to prevent VIDD in rodents using intermittent phrenic nerve stimulation and novel mitochondrial-targeted antioxidants (MTAs). 2) To further explore a novel mechanism of VIDD involving microRNA regulation of FOXOs, and to test the therapeutic potential of microRNAs. Phrenic nerve stimulation has never been evaluated in any model of VIDD, despite the fact that VIDD clearly results from diaphragm inactivity and that the phrenic nerve is anatomically accessible to stimulation in patients. We hope to establish proof-of-concept that phrenic stimulation will prevent VIDD, allowing subsequent scale-up to a workable clinical stimulation device. Regarding MTAs, a therapeutic benefit in VIDD has been suggested by only a single, perhaps flawed, study using a small mitochondria-targeted peptide. In order to confirm the mechanism of action of MTAs in this setting and provide an alternative MTA that may be more easily advanced to the clinic, we propose to attack mitochondrial oxidative stress and thus VIDD by both endogenous and exogenous approaches. Our endogenous approach will test for prevention of VIDD by MV experiments in mice that are engineered to overexpress the mitochondria-resident antioxidant thioredoxin-2 (Trx2). Our exogenous approach will include both the exogenous delivery of a TAT-Trx2 fusion protein and the delivery of the MTA drugs, SKQ1 and MitoQ. In this series of experiments, we will use our new model of mouse MV / VIDA - a model which will also allow future exploration of VIDD using additional genetic mouse models. Lastly, we will confirm our preliminary evidence that microRNAs, particularly microRNA-320a, effect VIDA by increasing muscle proteolysis via FOXO-mediated overexpression of the E3 ubiquitin ligases atrogin and MuRF1. We will subsequently test the therapeutic potential of systemic microRNA therapy against VIDA. As a whole, these experiments will both advance our understanding of the basic mechanisms underlying VIDA/VIDD and make substantial steps towards effective clinical therapies by testing these in animals.